Oxidants such as hydrogen peroxide (H2O2) are implicated in mediating a wide array of human diseases including atherosclerosis, cancer, and neurodegenerative diseases. Oxidants contribute to disease processes by causing damage to biomolecules and altering cellular metabolism. Key among the targets for oxidative damage are structural proteins and enzymes. In order to understand how oxidative stress can cause disease, it is important to discover which proteins become affected by oxidative stress, to what degree they are modified, and the functional consequences of the modifications. Previous studies on protein oxidation involved exposing purified proteins to a source of oxidants (e.g., iron/ascorbate) and measuring the extent and consequences of the damage. More recently, we have directed our efforts towards studying induction of protein oxidation in tumor cells exposed to chemotherapy drugs. Reports in the literature suggest that induction of apoptosis by chemotherapeutic agents is mediated by oxidants generated within the cells. We set out to determine whether proteins become oxidized in this process and, if so, whether oxidative modification of specific proteins contributes to the apoptotic process. Over the past year, experiments were carried out to identify specific proteins which might undergo oxidation during chemotherapy-induced apoptosis and H2O2-induced necrosis. Emphasis was placed on identifying mitochondrial proteins that might become modified since mitochondrial function is so central to the apoptotic process. Apoptosis was induced with the drug VP-16 and protein oxidation was assessed by measuring protein carbonyls (Western blot assay) and total methionine sulfoxide (in collaboration with investigators at the NHLBI, NIH). To date, we have found no evidence of increased protein oxidation during VP-16-induced apoptosis. There was also no induction of lipid peroxidation, and antioxidant compounds were ineffective at inhibiting VP-16-induced cell death. Our results provide evidence that apoptosis does not necessarily involve oxidative stress and does not require protein oxidation. This finding is significant because it challenges the current dogma which suggests that formation of intracellular oxidants is an integral part of the apoptotic process. The work is currently being prepared for publication in a peer-reviewed journal.